Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases.
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Mitotic double-strand break (DSB)-induced gene conversion at MAT in Saccharomyces cerevisiae was analyzed molecularly in mutant strains thermosensitive for essential replication factors. The processivity cofactors PCNA and RFC are essential even to synthesize as little as 30 nucleotides following strand invasion. Both PCNA-associated DNA polymerases delta and epsilon are important for gene conversion, though a temperature-sensitive Pol epsilon mutant is more severe than one in Pol delta. Surprisingly, mutants of lagging strand replication, DNA polymerase alpha (pol1-17), DNA primase (pri2-1), and Rad27p (rad27 delta) also greatly inhibit completion of DSB repair, even in G1-arrested cells. We propose a novel model for DSB-induced gene conversion in which a strand invasion creates a modified replication fork, involving leading and lagging strand synthesis from the donor template. Replication is terminated by capture of the second end of the DSB. (+info)
Studies on the interactions between human replication factor C and human proliferating cell nuclear antigen.
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Proliferating cell nuclear antigen (PCNA) is a processivity factor required for DNA polymerase delta (or epsilon)-catalyzed DNA synthesis. When loaded onto primed DNA templates by replication factor C (RFC), PCNA acts to tether the polymerase to DNA, resulting in processive DNA chain elongation. In this report, we describe the identification of two separate peptide regions of human PCNA spanning amino acids 36-55 and 196-215 that bind RFC by using the surface plasmon resonance technique. Site-directed mutagenesis of residues within these regions in human PCNA identified two specific sites that affected the biological activity of PCNA. Replacement of the aspartate 41 residue by an alanine, serine, or asparagine significantly impaired the ability of PCNA to (i) support the RFC/PCNA-dependent polymerase delta-catalyzed elongation of a singly primed DNA template; (ii) stimulate RFC-catalyzed DNA-dependent hydrolysis of ATP; (iii) be loaded onto DNA by RFC; and (iv) activate RFC-independent polymerase delta-catalyzed synthesis of poly dT. Introduction of an alanine at position 210 in place of an arginine also reduced the efficiency of PCNA in supporting RFC-dependent polymerase delta-catalyzed elongation of a singly primed DNA template. However, this mutation did not significantly alter the ability of PCNA to stimulate DNA polymerase delta in the absence of RFC but substantially lowered the efficiency of RFC-catalyzed reactions. These results are in keeping with a model in which surface exposed regions of PCNA interact with RFC and the subsequent loading of PCNA onto DNA orients the elongation complex in a manner essential for processive DNA synthesis. (+info)
Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin.
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Chromatin assembly factor 1 (CAF-1) is required for inheritance of epigenetically determined chromosomal states in vivo and promotes assembly of chromatin during DNA replication in vitro. Herein, we demonstrate that after DNA replication, replicated, but not unreplicated, DNA is also competent for CAF-1-dependent chromatin assembly. The proliferating cell nuclear antigen (PCNA), a DNA polymerase clamp, is a component of the replication-dependent marking of DNA for chromatin assembly. The clamp loader, replication factor C (RFC), can reverse this mark by unloading PCNA from the replicated DNA. PCNA binds directly to p150, the largest subunit of CAF-1, and the two proteins colocalize at sites of DNA replication in cells. We suggest that PCNA and CAF-1 connect DNA replication to chromatin assembly and the inheritance of epigenetic chromosome states. (+info)
The role of antiapoptotic Bcl-2 family members in endothelial apoptosis elucidated with antisense oligonucleotides.
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In this study, we utilized potent antisense oligonucleotides to examine the role of two Bcl-2 family members found in human umbilical vein endothelial cells (HUVEC). The first, A1, is thought to be a TNF-alpha-inducible cytoprotective gene, and the second, Bcl-XL, is constitutively expressed. Inhibition of the constitutive levels of Bcl-XL caused 10-25% of the cell population to undergo apoptosis and increased the susceptibility of cells to treatment with low concentrations of staurosporin or ceramide. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2 prevented DNA fragmentation and DeltaYm loss caused by Bcl-XL inhibition or Bcl-XL inhibition combined with staurosporin. However, disruption of DeltaYm caused by Bcl-XL inhibition combined with ceramide treatment was not inhibited by benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2, although DNA fragmentation was completely prevented. Taken together, these results demonstrate a direct protective role for Bcl-XL under normal resting conditions and under low level apoptotic challenges to HUVEC. Furthermore, Bcl-XL protects cells from caspase-dependent and -independent mechanisms of DeltaYm disruption. In contrast to Bcl-XL, A1 inhibition did not show a marked effect on the susceptibility of HUVEC to undergo apoptosis in response to TNF-alpha, ceramide, or staurosporin. These results demonstrate that although A1 may be a cytoprotective gene induced by TNF-alpha, it is not primarily responsible for HUVEC resistance to this cytokine. (+info)
Overexpression of A1, an NF-kappaB-inducible anti-apoptotic bcl gene, inhibits endothelial cell activation.
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A1 is an anti-apoptotic bcl gene that is expressed in endothelial cells (EC) in response to pro-inflammatory stimuli. We show that in addition to protecting EC from apoptosis, A1 inhibits EC activation and its associated expression of pro-inflammatory proteins by inhibiting the transcription factor nuclear factor (NF)-kappaB. This new anti-inflammatory function gives a broader dimension to the protective role of A1 in EC. We also show that activation of NF-kappaB is essential for the expression of A1. Taken together, our data suggest that A1 downregulates not only the pro-apoptotic and pro-inflammatory response, but also its own expression, thus restoring a quiescent phenotype to EC. (+info)
Survival activity of Bcl-2 homologs Bcl-w and A1 only partially correlates with their ability to bind pro-apoptotic family members.
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Certain Bcl-2 family members promote cell survival, whereas others promote apoptosis. To explore further how heterodimerization of opposing members affects survival activity, we have compared the abilities of the anti-apoptotic Bcl-w and A1 to bind to the pro-apoptotic Bax, Bak, Bad and Bik and to protect cells from their cytotoxic action. Bcl-w co-immunoprecipitated from cell lysates with Bax, Bak, Bad and Bik, but A1 bound only Bak and Bik. Mutation of A1 at a highly conserved glycine within the BH1 domain prevented binding, but the comparable Bcl-w mutant still bound Bak, Bad and Bik, indicating that the glycine is not essential for all heterodimerization. Bcl-w and A1 protected against apoptosis induced by over-expression of Bax or Bad but not that induced by Bak or Bik. With several gene pairs, binding and protection were discordant. The results may reflect critical threshold affinities but also suggest that certain pro-apoptotic proteins may also contribute to apoptosis by a mechanism independent of binding pro-survival proteins. (+info)
The murine antiapoptotic protein A1 is induced in inflammatory macrophages and constitutively expressed in neutrophils.
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Myeloid leukocytes are thought to regulate their susceptibility to apoptosis upon migration to a site of inflammation. However, factors that determine survival have not been well characterized in these cells. We have examined the expression of murine A1, an antiapoptotic Bcl-2 relative found in activated myeloid cells, during the course of an acute inflammatory response. Intraperitoneal infection of mice with the virulent RH strain of Toxoplasma gondii led to a 5- to 10-fold increase in A1 mRNA levels in peritoneal cells after several days. Bcl-2 expression was unchanged. The increase in A1 expression depended on the dose of the organism and coincided with a sharp increase in peritoneal cellularity. A1 protein levels were also increased as determined by Western blot analysis and immunohistochemical studies. All neutrophils and approximately half of the macrophages in the inflammatory exudate contained high levels of A1 in cytoplasm. A1 expression did not correlate with intracellular parasitization. Peripheral blood neutrophils from normal mice strongly expressed A1 protein, whereas normal monocytes showed only weak staining. Bax mRNA was induced in parallel with A1 in macrophages. Exudate macrophages and granulocytes that were apoptotic by TUNEL staining occasionally appeared to display A1 throughout the cell nucleus. These studies identify A1 as a potential regulator of apoptosis during acute inflammation. (+info)
Electron microscopic analysis reveals that replication factor C is sequestered by single-stranded DNA.
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Replication factor C (RF-C) is a eukaryotic heteropentameric protein required for DNA replication and repair processes by loading proliferating cell nuclear antigen (PCNA) onto DNA in an ATP-dependent manner. Prior to loading PCNA, RF-C binds to DNA. This binding is thought to be restricted to a specific DNA structure, namely to a primer/template junction. Using the electron microscope we have examined the affinity of human heteropentameric RF-C and the DNA-binding region within the large subunit of RF-C from Drosophila melanogaster (dRF-Cp140) to heteroduplex DNA. The electron microscopic data indicate that both human heteropentameric RF-C and the DNA-binding region within dRF-Cp140 are sequestered by single-stranded DNA. No preferential affinity for the 3' or 5' transition points from single- to double-stranded DNA was evident. (+info)